Hybrid strip

ABSTRACT

A method of determining concentrations of a plurality of analytes from a single blood sample placed in a single opening. A portion of the single blood sample is absorbed by a test matrix that includes a plurality of layers and a chromogenic agent. A colored response is generated by the test matrix. The colored response is proportional to the concentration of a first analyte. A portion of the single blood sample is drawn into a capillary tube and placed in contact with an electrode and a counter-electrode. An electrical property of the single blood sample is analyzed through the electrode and counter-electrode. The electrical property is proportional to the concentration of a second analyte in the single blood sample.

BACKGROUND OF THE INVENTION

The level of certain analytes in blood and other body fluids is oftenused to diagnose disease, determine disease risk factors, monitor thecourse of a therapy, or determine the presence of illicit drugs. Inrecent years, analytes carried in blood have been evaluated to determinevarious cholesterol and triglyceride levels as a significant indicatorof risk of coronary heart disease. In managing heart disease, physicianscommonly order what is referred to in the art as a “full lipid panel”for patients to determine the concentration of total cholesterol, highdensity lipoprotein cholesterol (HDL), low density lipoproteincholesterol (LDL), and triglycerides. Glucose and ketone dry hybrid teststrips are used for managing diabetes. Ketone hybrid test strips alsoare useful in managing weight loss. Hybrid test strips for determiningcreatinine concentration in the blood or other bodily fluids are usedfor diagnosing and treating impaired kidney function and a variety ofother metabolic disorders and diseases.

While clinical tests have been used and still are being used todetermine the concentration of the above-mentioned analytes, more andmore physicians and consumers are relying on dry hybrid test strips foreconomical and easier measurement, particularly when testing at shorterintervals, such as days or weeks, is important or when rapid results arecritical. Furthermore, for such tests to be practical for consumers andphysicians, the devices used for testing must be small and portable. Forcertain users, diabetics testing for glucose levels for example,portability is key since glucose levels must be frequently tested tomaintain proper insulin levels. As a result of this need, numerous smalland portable devices have been developed to test for analytes.

The mechanisms used in determining the levels of analytes in the bloodfall into a number of categories, including but not limited to:photometric, electrchemical (ampherometric and coulmetric), andpotentiometric. Photometric blood testing typically involves reacting ablood sample with a reagent, shining a light on the reacted sample, andmeasuring the light reflected. Electrochemical blood testing involvesreacting the blood with a reagent, subsequently applying an excitationvoltage to the reacted sample, and measuring the effect of theexcitation voltage. Potentiometric testing involves measuring thepotential (or voltage) using analyte specific electrodes.

Presently available devices perform tests according to one of theabove-mentioned mechanisms. Since the mechanisms require significantlydifferent systems, to keep devices small, an omnibus device has not beencreated. Furthermore, many users only need to test for a single analyteand, therefore, a system performing tests according to a singlemechanism would meet their needs.

Diabetes and heart disease are “silent killers” that affect more than200 million Americans. Since 1900, heart disease has been the leadingcause of death in the United States, costing the health care system morethan $326.6 billion annually. Risk of heart disease or stroke is 2 to 4times greater for people with diabetes than for the average person. Ofpeople receiving diagnoses of type 2 diabetes, 50% are not aware oftheir risk until after their first heart attack or stroke. For most ofthe patients with type 2 diabetes, an average of 7 to 10 years elapsesfrom the time that heart disease develops to its diagnosis.

BRIEF SUMMARY OF THE INVENTION

Since many individuals who suffer from diabetes also are at risk forheart disease, such individuals or their physicians may desire that theyregularly test not only for glucose levels related to their diabeticcondition, but also for various cholesterol and triglyceride levels as asignificant indicator of risk of coronary heart disease. Presently, auser is unable to run both an electrochemical test yielding glucoselevels and a photometric test yielding various cholesterol andtriglyceride levels with a single device and a single strip. Not onlywould a user need to have two separate devices, the user would need totake two separate blood samples. The time required to perform two testsand the need to take two blood samples discourages many individuals fromroutinely performing such tests as needed or directed by their doctor.

Furthermore, some individuals suffer from conditions that make certaintest mechanisms for analytes ineffective. Examples of some of theseconditions include, but are not limited to: sickle-cell disease,end-stage renal disease, or other conditions that alterhemoglobin/hematocrit (dehydration, anemia). Individuals with suchconditions must use technologies that minimize the interference fromhematocrit levels. This typically requires electrochemical testingtechniques. At the same time, electrochemical testing techniques do notallow the individual to test for certain analytes; they may onlyefficiently be tested by using photometric techniques.

A solution to the above problems is offered by providing a hybrid stripand device for utilizing the hybrid strip that allows for the testing ofmultiple analytes by a variety of methods. These methods includephotometric testing methods and electrochemical testing techniques,including coulmetric, potentiometric, and amphermetric techniques.

In one embodiment of a method of determining concentrations of aplurality of analytes from a single blood sample, placed in a singleopening, a hybrid test strip includes electrochemical and photochemicaltesting mechanisms to determine the concentration of various analytes. Aportion of the single blood sample is absorbed by a test matrix thatincludes a plurality of layers and a chromogenic agent. A coloredresponse is generated by the test matrix. The colored response isproportional to the concentration of a first analyte. A portion of thesingle blood sample is drawn into a capillary tube and placed in contactwith an electrode and a counter-electrode. An electrical property of thesingle blood sample is analyzed through the electrode andcounter-electrode. The electrical property is proportional to theconcentration of a second analyte in the single blood sample.

In one embodiment of a method of determining concentrations of aplurality of analytes from a single blood sample using a hybrid teststrip, placed in a single opening, the single blood sample is contactedwith the top surface of an elongated disbursement layer. The sample isspread substantially throughout the entire length of the disbursementlayer. The single blood sample is contacted with the end of a capillarytube, such that a portion of the single blood sample is drawn into thecapillary tube. The blood sample is delivered from the disbursementlayer to a first stack, a second stack, and a third stack, each of thefirst, second, and third stacks positioned adjacent to and in constantcontact with the disbursement layer. The sample is moved downwardthrough the stacks including through the blank layer in a directionsubstantially normal to the plane defined by the stacks. A coloredresponse is produced at the bottom of each of the three stacks, thecolored response at the bottom of the first stack being proportional tothe concentration of a first analyte in the blood sample, the coloredresponse at the bottom of the second stack being proportional to theconcentration of a second analyte in the blood sample, and the coloredresponse at the bottom of the third stack being proportional to theconcentration of a third analyte in the blood sample. A portion of thesingle blood sample drawn into the capillary tube is delivered to anelectrode and a counter-electrode. An electrical property of the singleblood sample is measured using the electrode and counter-electrode. Theelectrical property is proportional to the concentration of a fourthanalyte in the blood sample. In one alternative, the sample chamberfurther includes a loosely woven material that assists in the flow ofthe sample to the electrodes. In this alternative, the sample chambermay not utilize a capillary effect. In another alternative, the samplechamber may also not utilize the capillary effect but instead thepermissively of the spreading layer is sufficient to spread the sampleinto the sample chamber.

In an embodiment of an apparatus for measuring concentration of multipleanalytes in a whole blood sample, a hybrid test strip is used. Theapparatus includes a test matrix having an elongated porous disbursementlayer, a blood separation layer adjacent to the bottom surface of thedisbursement layer, and at least two vertically aligned stacks spacedapart and adjacent to the bottom surface of the blood separation layer.A first one of the vertically aligned stacks includes multiple layers,the multiple layers including a reagent and a chromagen. The apparatusfurther includes an electrochemical testing member that includes acapillary tube having a first end and a second end, an electrode, and acounter-electrode. The electrode and counter-electrode are oriented inelectrical communication with each other when a blood sample is present.A hybrid test strip holder having top and bottom portions sandwiches thetest matrix between the top and bottom portion. The top and bottomportions hold the electrochemical testing member. The top portion of thehybrid test strip holder has a sample application window exposing a topsurface of the disbursement layer and the first end of the capillarytube. The bottom portion of the hybrid test strip holder has at leastone test reading window through which bottom surfaces of the first andsecond stacks can be read.

An embodiment of a method of using a hybrid test strip for determiningconcentrations of a plurality of analytes from a single blood sample,placed in a single opening, includes separating a top portion of ahybrid test strip holder from the bottom portion of a hybrid test stripholder. An electrode chemical testing member is placed on the bottomportion of the hybrid test strip holder such that a hole in theelectrode chemical testing member is on a rod of the bottom portion. Thetop portion of the hybrid test strip holder and the bottom portion ofthe hybrid test strip holder are connected such that the electrochemicaltesting member is sandwiched between. The hybrid test strip holderhouses a test matrix that includes an elongated porous disbursementlayer, a blood separation layer adjacent to the bottom surface of thedisbursement layer, and at least two vertically aligned stacks spacedapart and adjacent to the bottom surface of the blood separation layer,wherein a first one of the vertically aligned stacks includes multiplelayers, the multiple layers including a reagent and a chromagen.

One embodiment of a method of determining concentrations of a pluralityof analytes from a single blood sample, placed in a single opening,includes placing the single blood sample in an opening; absorbing theblood sample with a test matrix that includes a plurality of layers anda chromogenic agent; generating a colored response with the test matrix,wherein the colored response is proportional to the concentration of afirst analyte; drawing a portion of the single blood sample into acapillary tube; contacting the portion of the single blood sample withan electrode and a counter-electrode; and measuring an electricalproperty of the single blood sample though the electrode andcounter-electrode, wherein the electrical property is proportional tothe concentration of a second analyte in the single blood sample. Onefeature of the method is that the drawing may be accomplished by acapillary effect of the capillary tube.

In another embodiment, a method of determining concentrations of aplurality of analytes from a single blood sample, placed in a singleopening, includes: contacting the single blood sample with the topsurface of an elongated disbursement layer and spreading the samplesubstantially throughout the entire length of the disbursement layer;contacting the single blood sample with the end of a capillary tube,such that a portion of the single blood sample is drawn into thecapillary tube; delivering the blood sample from the disbursement layerto a first stack, a second stack, and a third stack, each of the first,second, and third stacks positioned adjacent to and in constant contactwith the disbursement layer, and moving the sample downward through thestacks in a direction substantially normal to the plane defined by thestacks; producing a colored response at the bottom of each of the threestacks, the colored response at the bottom of the first stack beingproportional to the concentration of a first analyte in the bloodsample, the colored response at the bottom of the second stack beingproportional to the concentration of a second analyte in the bloodsample, and the colored response at the bottom of the third stack beingproportional to the concentration of a third analyte in the bloodsample; delivering the portion of the single blood sample drawn into thecapillary tube to an electrode and a counter-electrode; and measuring anelectrical property of the single blood sample using the electrode andcounter-electrode, wherein the electrical property is proportional tothe concentration of a fourth analyte in the blood sample.

One feature of the method is that at least one of the stacks includes ablank layer, the blank layer being a different layer than thedisbursement layer. The blank layer primarily functions to maintain allstacks at substantially the same thickness. Another feature may includethat the first, second, and third analytes are Total Cholesterol, HDLCholesterol, and Triglycerides respectively. Another feature may includethat the fourth analyte is glucose. Another feature may include aselectively permeable membrane introduced between the electrode andcounter-electrode in order to lessen interferents. Another feature mayinclude a selective electrocatalyst introduced in order to lesseninterferents.

An embodiment of an apparatus for measuring a concentration of multipleanalytes in a whole blood sample includes a test matrix, anelectrochemical testing member, and a hybrid test strip holder havingtop and bottom portions sandwiching the test matrix therebetween and thetop and bottom portions holding the electrochemical testing member, thetop portion of the hybrid test strip holder having a sample applicationwindow exposing a top surface of a disbursement layer and a first end ofa capillary tube, and a bottom portion of the hybrid test strip holderhaving at least one test reading window through which bottom surfaces ofthe first and second stacks can be read. The test matrix includes anelongated porous disbursement layer; a blood separation layer adjacentto the bottom surface of the disbursement layer; and at least twovertically aligned stacks spaced apart and adjacent to the bottomsurface of the blood separation layer wherein a first one of thevertically aligned stacks includes multiple layers, the multiple layersincluding a reagent and a chromagen. The electrochemical testing memberincludes a capillary tube having a first end and a second end; anelectrode; and a counter-electrode, wherein the electrode andcounter-electrode are oriented in electrical communication with eachother when a blood sample is present.

A feature of the apparatus includes that the sample application windowis positioned within a periphery defined by the stacks. Another featureincludes that the bottom surfaces of the stacks are substantiallycoplanar. Another feature includes that the blood separation layercomprises a glass fiber matrix. Another feature includes that theelectrochemical test panel includes enzymatic reactants overlaying theelectrode and counter-electrode. Another feature includes that aselectively permeable membrane is introduced between the electrode andcounter-electrode in order to lessen interferents. Another featureincludes that a selective electrocatalyst is introduced in order tolessen interferents. Another feature includes that the electrochemicaltesting member is interchangeable.

In an embodiment of a method of customizing a hybrid test strip fordetermining concentrations of a plurality of analytes from a singleblood sample, placed in a single opening, the method includes separatinga top portion of a hybrid test strip holder from the bottom portion of ahybrid test strip holder, the bottom portion of the hybrid test stripholder having a rod; placing an electrode chemical testing member on thebottom portion of the hybrid test strip holder such that a hole in theelectrode chemical testing member is on the rod of the bottom portion;and connecting the top portion of the hybrid test strip holder and thebottom portion of the hybrid test strip holder such that theelectrochemical testing member is sandwiched between. In thisembodiment, the hybrid test strip holder houses a test matrix includingan elongated porous disbursement layer, a blood separation layeradjacent to the bottom surface of the disbursement layer, and at leasttwo vertically aligned stacks spaced apart and adjacent to the bottomsurface of the blood separation layer. A first one of the verticallyaligned stacks includes multiple layers, the multiple layers including areagent and a chromagen. A feature of an embodiment includes thecapability to tailor the hybrid strip to test for a variety of analytesaccording to the optimal testing methodology.

In another embodiment as system for sample retention using a test striphaving a body. Within the body is a spreader layer configured to evenlyspread the fluid sample received through the port. Multiple sampleretention areas and test areas may be included in the body of the teststrip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an embodiment of a hybrid strip;

FIG. 2 is a perspective view of an embodiment of a hybrid strip wherethe top portion has been removed;

FIG. 3 is a perspective view of an alternative embodiment of a hybridstrip where the top portion has been removed;

FIG. 4 is an exploded perspective view of a hybrid strip showing thelayers of a test matrix;

FIG. 5 is cross-section view of a test matrix;

FIG. 6 is a perspective view illustrating a vertical flow schemeutilized by stacks;

FIGS. 7-9 depict various embodiments of “vertically aligned” layers;

FIG. 10 is a top view of an electrochemical testing member;

FIG. 11 is a top view of a hybrid strip;

FIGS. 12-14 illustrate standard curves for cholesterol, HDL, andtriglycerides, respectively;

FIGS. 15-17 are graphs which plot measured value of concentration versusreference value for HDL, total cholesterol, and triglycerides,respectively; and

FIGS. 18-20 illustrate standard curves for cholesterol, HDL, andglucose, respectively.

DETAILED DESCRIPTION

The embodiments described below are not intended to be exhaustive or tobe limited to the precise forms disclosed in the following detaileddescription. Rather, the embodiments are chosen and described so thatothers skilled in the art may appreciate and understand the principlesand practices disclosed herein.

Definitions

“HDL” refers to high density lipoprotein.

“LDL” refers to low density lipoprotein.

“VLDL” refers to very low density lipoprotein.

“NonHDL” refers to LDL, VLDL, and chylomicrons, as well as lipoproteinsother than HDL that will react with a conventional cholesterol reactionmembrane.

“PTA” refers to phosphotungstic acid.

“HDL fractionation layer” refers to a dry hybrid test strip layerselected from suitable materials and impregnated with one or morereagents such that non-HDL cholesterol (VLDL and LDL) in a fluid sampledeposited on the layer are both precipitated and substantially retainedwithin the layer, but HDLs in solution in the sample remain in solutionand are able to pass through the fractionation layer.

“Plasma” refers to the non-cellular portion of blood from which cellularcomponents such as red blood cells are excluded.

“Serum” technically differs from plasma in that it does not includefibrinogen. However, for purposes of this application, “serum” and“plasma” are sometimes used interchangeably.

Referring to FIG. 1, hybrid test strip 20 includes hybrid test stripholder 22. Hybrid test strip holder 22 may be formed of a variety ofdifferent materials including, but not limited to, metal and plastic. Inthe case of a plastic hybrid test strip holder 22, the plastic may beformed using injection molding. The hybrid test strip holder 22 includesan opening 32 for receiving a blood sample. Opening 32 is depicted as anoval-shaped opening in FIG. 1; however, alternative openings of alltypes of geometric shapes are possible including circles, squares,rectangles, etc. Handle 24 is depicted in FIG. 1, allowing for a user toconveniently handle the hybrid test strip using Handle 24. Handle 24 isan optional feature, however, and need not be included for the functionof hybrid test strip 20. Hybrid test strip 20 includes anelectrochemical testing member 100 that is integrated with thephotochemical portion.

As depicted in FIG. 2, hybrid test strip 20 includes a top portion 26and a bottom portion 30. The top portion includes opening 32 and thebottom portion 30 includes openings 34. Although in FIG. 2 threeopenings 34 are shown, various configurations of openings are possible.The openings 34 provide for a window for photometric testing of samples.Generally, the photometric testing through openings 34 is a measure ofreflectance; however, other photometric testing techniques may be usedincluding transmissivity and absorption. The number of openingscorresponds to the number of samples that are subjected to photometrictesting. Optionally, openings 34 can be configured with transparentwindows.

Top portion 26 and bottom portion 30 fit together as shown in FIG. 2. Avariety of configurations are possible for fitting the top portion 26and the bottom portion 30 together. As shown in FIG. 2, side bars 28, 29serve to hold the top portion 26 in place. Snap pieces 51 may be snappedon to side bars 28, 29 at grooves 52. This configuration provides afriction fit. Top portion 26 may be further secured with adhesive orsimply a friction fit. In one alternative, the top portion 26 and thebottom portion 30 may be hinged or non-hinged. The non-hinged end of thetop portion 26 may be held in place by a variety of locking mechanismsthat will be apparent to those skilled in the art in light of thisdisclosure.

In one alternative, shown in FIG. 3, the top portion 26 and the bottomportion 30 include a number of receptacles 50 housing a number of pegs49 that fit via a friction fit into mating cylindrical openings 56. Thistype of locking mechanism keeps the distance and fit of the top portion26 and the bottom portion 30 constant. An improper fit between the topportion 26 and the bottom portion 30 may cause irregular sample flow andresult in erroneous analyte measurements due to changes in the diffusionspeed and/or pattern of samples.

Hybrid strip 20 is designed to provide many different types of analytetesting. A first embodiment of the hybrid strip 20 includes two types ofanalyte testing, photometric and electrochemical. To support photometrictesting, hybrid strip 20 is configured with a test matrix 36 thatincludes at least one stack 42. In FIG. 4, an exploded view of testmatrix 36 is shown. Test matrix 36 is made up of a top disbursementlayer 38, a blood separation layer 40, stacks 42, and adhesive layer 44having openings 46 that align with openings 34 and the bottoms ofrespective stacks 42 when the layers are assembled. Stacks 42 arefurther made up of one or more vertically aligned layers, the functionand specifics of which are described in further detail below. Whenassembled and closed, the layers of stacks 42 and layers 38, 40, and 44are all pressed together. Opening 32 exposes a part of the top surfaceof disbursement layer 38, and openings 34 and 46 expose the bottomsurface of bottom portion 30 and the bottom layers of stacks 42.

In another embodiment, the test strip may include multiple differenttest areas, retention areas, and/or distribution areas (togetherreferred to as test elements and the strip referred to as a multi-testelement strip. Test areas may include as mentioned about electrochemicaland photometric test areas and further may include other type of testareas, including liquid test areas, gel test areas, and gas interfacetest areas. Retention areas may include areas established for holdingblood for a set period of time before testing and areas for capturing asample of blood for later usage. Distribution areas may include areas towhich a testing device may be hooked up to siphon a portion of thesample for testing within a testing device.

A test strip with multiple different test areas, retention areas, and/ordistribution areas (together referred to as test elements) configurationgenerally has a spreading layer. The spreading layer functions to spreadthe sample so that it can reach the various test area, retention areas,and distribution areas that are included in the strip. The time ofabsorption for the spreading layer set in proportion to the amount ofspreading desired. The time of absorption may be modified by treatingthe spreading layer with materials impervious to liquid and by changingthe weave of the spreading layer. More spreading will be desired basedon the number test elements in the strip. Also the size of the sampleanticipated may limit the number of test elements that may be included.

Continuing with the above multi-test element strip, sample absorbed intothe spreading layer is generally be used to bring a sample to a teststack, composed of testing layers. The testing layers may ultimately beanalyzed based on photometric or electrochemical techniques.Alternatively, absorbed sample may be transferred to a gel testing area.During spreading (and absorption), part of the sample may be captured bycapillary tubes or other mechanisms, such at absorbent materials. Theseportions of the sample may be transferred to other test elements. In oneexample, that is explained further herein, a capillary tube carries aportion of the sample to be tested electrochemically. Alternatively, thesample may be deposited in a storage retention area. This storageretention area is designed to keep a historical sample record and may besealed by the user depressing a tab on the test strip that closes offthe storage retention area. Alternatively, a delay retention area may beused. The sample portion may be transferred to the delay retention areausing the above described techniques. In the delay retention areaincludes a slow absorbing layer that slows the process of the sampleportion to the test area. Such a technique may be used to test for theoxygen absorption rate of a sample, where one sample is testedimmediately and another is delayed by the delay retention area. Such adelay retention area may be used whenever timing is important intesting. Alternatively, a portion of the sample may be transferred to adistribution area. When the test strip is inserted into a test device,sample may be transferred to the distribution area, for example by avacuum suction. Due to the usage of the spreading layer a test strip maybe configured with numerous alternative test elements.

Continuing with the hybrid strip example, referring to FIG. 5, theindividual layers and the diagnostic chemistries of matrix 36 can beappreciated. The top layer 38 of matrix 36 is a disbursement or spreaderlayer capable of efficiently spreading the blood sample 58 through itsentire length such that the blood sample 58 is deposited vertically tothe next layer over the entire length of layer 38 (see reference arrowsin FIG. 5). For example, a mesh, such as polyester mesh, works well forsingle hybrid test strips, such as those disclosed in U.S. Pat. No.5,597,532. However, when such mesh is used in an attempt to spread bloodacross an elongated matrix such as matrix 36, the blood inevitably isdrawn to the layer below the mesh (layer 40) before it spreads to theouter ends of layer 38. If this occurs, no blood will be drawn into theelectrochemical testing member 100. Therefore, a lower permissivelyspreading layer may be required to integrate the testing mechanisms.

The problem of blood being drawn into layer 40 from layer 38 presented aserious design hurdle. The problem is caused in large part by layer 40,which is a glass fiber depth filter that is adjacent to and in contactwith layer 38. When in contact with layer 38, layer 40 exerts a wickingeffect on layer 38, tending to draw blood into layer 40 at its centerbefore the blood can sufficiently spread to the ends of the elongateddisbursement layer 38. A sufficient blood sample is delivered to themiddle of layer 40, but not to its ends. This results in unpredictableand uneven deposition of the blood filtrate onto stacks 86 and 98 andcan result in blood not being drawn into the electrochemical testingmember 100, which in turn results in unpredictable test results.

Remarkably, the disbursement or spreader layer 38 spreads blood sample58 (FIG. 5) efficiently and sufficiently throughout the entire length oflayer 38 as shown by the reference arrows—even with layer 40 being inconstant contact therewith. This is a significant achievement, in thatit allows a multi-analyte dry phase hybrid test strip that uses only asingle 35 microliter sample of blood yet has no moving parts.

Without wishing to be tied to any specific theory, it is believed thatlayer 38 operates by a two-stage mechanism, although it should beunderstood that the steps may not occur sequentially, but instead mayoccur simultaneously to a certain degree. In the first step, bloodsample 58 (FIG. 5) spreads laterally within layer 38; in the secondstep, the sample is deposited vertically onto layer 40. Again, it shouldbe expected that the second step may begin at the central portion oflayer 38 before it occurs at the ends of layer 38; but there areinarguably two functions occurring, the first being spreading the bloodsample throughout the entire length of layer 38, and the second beingdelivering the blood sample uniformly to the next layer over the entirelength of layer 38.

Surprisingly, it has been found that layers used as conjugate pads inpregnancy test kits perform quite well as layer 38. Layer 38 is an opencell layer capable of rapidly and effectively spreading the fluidsample. One suitable material for layer 38 is available under the nameAccuflow Plus-P available from Schleicher & Schuell Bioscience, Inc.,Keene, N.H. Another suitable material for layer 38 is available underthe name Accuwik™ manufactured by Pall Corporation, East Hills, N.Y.Layer 38 may be constructed of hydroxylated polyester. The fibersurfaces have been modified to be inherently and permanentlywater-wettable. Layer 38 provides an excellent wicking rate and highvolume retention capability, which allows the blood to spread laterallyacross the entire length of the layer. U.S. patent application Ser. No.10/663,555 entitled “Test Strip And Method For Determining LDLCholesterol Concentration” filed on Sep. 16, 2003, and published Jul. 1,2004 under US Patent Application Publication No. 2004/0126830 is herebyincorporated by reference.

Generally, layer 38 must provide extremely consistent flowcharacteristics, be intrinsically water-wettable, and exhibit sufficientvolume retention capability such that the sample spreads throughout theentire length of the layer, even though another layer such as layer 40that acts as a wick is positioned in constant contact therebelow. It isanticipated that other layers possessing the above characteristics wouldwork for layer 38. Furthermore, see the discussion below of thenecessity to enable flow to the electrochemical testing member 100.

As will become clearer with reference to the discussion below,substantial lateral spreading occurs only in disbursement layer 38 ofmatrix 36. In the remaining layers, the net direction of fluid flow isbelieved to be substantially vertical, or normal, to the plane of thelayers. For example, with reference to FIG. 7, fluid sample drop 60 isdeposited onto layer 62 (which could be blood separation 10 on layer 40or one of the layers from one of stacks 42). Layer 62 defines a plane 64that is substantially parallel therewith. Transfer of fluid throughlayer 62 is normal or perpendicular to plane 64, or in the direction ofvector V, shown at reference numeral 66. Thus, there is no substantialmigration of fluid from one side of layer 62 to the other. Fluid flow isthrough layer 62, not across it.

In this connection, it should also be appreciated that, even thoughlateral spreading of a fluid sample occurs in layer 38, the sampleapplication window 32 is substantially vertically aligned with or atleast partially projects over the test reading windows 34 as shown inFIG. 4. The length of hybrid test strip 20 is governed by the peripheraldimension of the stacks 42. As shown in FIG. 4, test stacks 42 define alengthwise periphery “P”, whereas test application window 32 defines alengthwise periphery “p”. The test window 32 can be positioned withinthe periphery P defined by stacks 42 as shown in FIG. 4. This allows amore compact hybrid test strip than in a lateral flow device, whereintest window 32 would be positioned outside of the lengthwise peripheryP, thus requiring a longer strip.

Furthermore, because lateral flow does not occur in any of the layersother than layer 38, the layers can be “vertically aligned”, as shown inFIGS. 7-9. With particular reference to FIG. 7, equal size layers 68,70, and 72 of stack 74 are aligned directly over one another. While suchdirect alignment may be advantageous because it is most compact, itshould be understood that other minor variations of vertical alignmentare within the scope of this disclosure. For example, FIG. 9 depicts astack 76 in which middle layer 80 is larger than and protrudes slightlyfrom layers 78 and 82. Similarly, stack 84 shown in FIG. 9 is depictedas crooked, wherein the layers thereof are not placed directly over oneanother.

The hybrid strip 20 has an integrated electrochemical testing mechanism.The ability of the hybrid strip to perform both sets of tests is due inpart to the initial spreading of the sample, such that theelectrochemical testing member can receive a sufficient sample fortesting. Electrochemical blood testing generally refers to a procedurewhere the resistance, potential (charge), or voltage of a sample ismeasured, and includes coulometric, voltammetric, ampherometric, andpotentiometric techniques. In coulometry, the amount of mattertransformed during an electrolysis reaction is determined by measuringthe amount of electricity (in coulombs) consumed or produced. The amountof electricity produced may be correlated to the analyte concentration.In voltammetry, information about an analyte is obtained by measuringthe current as the potential is varied. In ampherometry, the resistanceof an undercurrent flow is measured and this resistance is correlated toanalyte concentration. In potentiometry, the potential of a sample underno current flow is measured. The measured potential then may be used todetermine the analytical concentration of some components of the analytegas or solution. In the present application, although theelectrochemical mechanism employed by the hybrid strip 20 may bedescribed in relation to a specific testing technique, any of thesetesting techniques may be utilized with small modifications that will beapparent to those skilled in the art in light of this disclosure.

Referring to FIGS. 9 and 10, electrochemical testing member 100, shownin FIG. 5, is designed to fit between top portion 26 and bottom portion30 such that the end of testing member 100 extends beyond the area ofthe hybrid test strip defined by top portion 26 and bottom portion 30.The electrochemical testing member 100 has a sample chamber 110 intowhich the blood sample to be tested is transferred. Electrode 115 andcounter-electrode 120 extend into sample chamber 110. Sample chamber 110is configured such that electrode 115 and counter-electrode 120 will beimmersed in the sample. Sample chamber 110 may be a tube with capillaryproperties. When a drop of blood is placed in window 32, the bloodspreads across disbursement layer 38. When the blood comes into contactwith sample chamber 110, the capillary effect pulls some of the sampleinto sample chamber 110. The sample chamber 110 receives blood by virtueof the initial spreading, which allows a test sample area to receivesufficient blood to perform the required testing. In one alternative,the initial permissivity of the spreading layer (layer 38) is reducedsuch that sufficient blood can reach the sample chamber. A cap 130 fitsover electrode 115, counter-electrode 120, and sample chamber 110resulting in an enclosed tested area, open only at the point at whichthe sample deposited in window 32 enters.

In one embodiment, in order for the spreading layer (layer 38) tosufficiently spread the blood initially, but then allow absorption, thelayer may be treated or woven more tightly to enable initial spreading.In an alternative embodiment, treatment may include adding sorbitanmonooleate and/or polyethoxylated hydrogenated castor oil with a lowpercentage coating based on weight. In one embodiment, the coating levelmay be 0.01-0.1%. The coating level may be based at least in part on thesurface area of the spreading layer; the larger the surface area, themore of a coating will be needed in order to allow for initial spreadingto the electrochemical testing member.

In one embodiment, the sample chamber 110 may include a loosely wovenmaterial that quickly absorbs liquid in contrast to the spreading layer.The electrodes may be positioned in the loosely woven material such thatwhen the material absorbs the sample, the electrodes are in contact withthe sample. In one alternative, the loosely woven material iselectrochemically neutral and does not greatly affect theelectrochemical testing procedure. The loosely woven material mayinclude loosely formed cellulosic fibers and may also containsuperabsorbent polymer fibers to improve the absorbent capacity. Theterm “superabsorbent polymer” (or “SAP”) generally refers to materialswhich are capable of absorbing and retaining at least about 10 timestheir weight in body fluids under a 0.5 psi pressure. Suchsuperabsorbent polymers include crosslinked hydrophilic polymers, suchas polyvinyl alcohols, polyethylene oxides, crosslinked starches, guargum, xanthan gum, and the like. One example of a commercially availableSAP material is AQUAKEEP SA-70, available from Sumitomo Seika ChemicalsCo., Ltd. In usage, the loosely woven material may allow for the samplechamber to be larger and less bounded since the loosely woven materialmay assist in the flow of the sample. In yet another embodiment, thesample chamber 110 may also not employ the capillary effect, but insteadsimply rely to the ability of the spreading layer to resist thepenetration of the sample sufficiently for the sample to spread into thesample chamber 110.

Connections 121, 122 extend from electrode 115 and counter-electrode 120to leads 123, 124. Referring to FIG. 11, opening 125 is designed to fitonto rod 49 to allow for a friction fit for the electrochemical testingmember 100. Electrochemical testing member 100 is also held in place bytop portion 26 and bottom portion 30.

In the pictured embodiment, electrode 115 and counter-electrode 120 areformed such that counter-electrode 120 substantially surrounds electrode115; however, such precise configuration is not required. The electrodesmay be oriented in a variety of arrangements, as long as they are inelectrolytic contact with the sample. In one alternative, electrode 115and counter-electrode 120 are separated by a distance of 0.01-1 mm. Thedistance separating the electrodes and the sample size may impact theprecision of analyte measurements.

For the electrodes to measure an analyte, a reagent must be introduced.A layer of enzymatic reactants overlays the electrodes and provides asubstrate for the sample. The reactant used is dependent on the type ofelectrochemical test being conducted and the analyte being tested for.Some examples of enzymatic reactants are described below. A variety ofreagents will be apparent to those skilled in the art in light of thisdisclosure.

To perform testing, the hybrid test strip is inserted into a testingdevice with optical and electrochemical testing mechanisms. Opticaltesting mechanisms involve a light source and a light sensor. The lightsensor senses the light reflected from the samples through openings 34.The measured light level is processed by an analog-to-digital (A/D)converter and fed to a microprocessor that performs the calculation todetermine the corresponding analyte level. The electrochemical testingmechanism is performed according to a similar procedure. The electricalproperty corresponding to the electrochemical method is tested for(voltage, resistance, current) and then converted by an A/D converterand processed by a microprocessor to determine the corresponding analytelevel.

The leads 123, 124 of the electrochemical testing member 100 aredesigned to line up with leads inside of the testing device such thatthe needed electrical stimulus can be applied and the result measured(in some cases, no electrical stimulus is applied). This measurement isconverted by an ND converter and processed by a microprocessor todetermine the corresponding analyte level.

Users receive the hybrid testing strip 10 as a complete testing strip,requiring the user to place a blood sample in window 32 and place thehybrid testing strip 10 into a testing device. In one alternative, thehybrid testing strip comes completely assembled and the analytes it isconfigured to test for is preset. In another alternative, theelectrochemical testing member 100 is interchangeable. In thisalternative, a hybrid strip 10 with preset photochemical testing stacksmay receive an electrochemical testing member selected by the user.Therefore, at the time of usage the user may customize the hybrid stripto receive a single blood sample and test for multiple analytes.

The electrochemical testing member 100 may be inserted by separating topportion 26 and bottom portion 30 (which may be hinged, see above) andfitting opening 125 on to rod 49. Alternative insertion and lockingtechniques may be utilized, which will be apparent to those skilled inthe art.

One embodiment of the present invention includes a lipid panel plusampherometric glucose. The lipid panel tests for Total Cholesterol, HDLCholesterol, and Triglycerides. The enzymatic reactions for the lipidpanel may be characterized as shown below:

HDL Measurement Stack

With reference to FIG. 5, middle stack 92 having layers 94 and 96 isadjacent to and in fluid communication with the bottom side of layer 40.Stack 92 takes fluid from layer 40 and produces a colored response inreaction layer 90 that is proportional to the concentration of HDLcholesterol. Layer 40 does not separate 100% of red blood cells.Instead, about 20% of red blood cells escape to layers 88, 94, and 100.Thus, layers 88, 94, and 100 separate and retain residual blood cellspassed to them from layer 40.

As noted above, the prior art generally teaches that two layers and twoassociated process steps are necessary to precipitate and separatenon-HDLs from plasma. According to the prior art approach, precipitationof non-HDLs is carried out in the first layer, and the precipitants thenpass through this first layer to a second layer. In the second layer,the precipitants' migration is slower than that of plasma, and theplasma reaches the test membrane before the precipitants. See, e.g., USPat. Nos. 5,426,030; 5,580,743; 5,786,164; 6,171,849; 6,214,570;5,451,370; 5,316,916; 5,213,965; and 5,213,964. In contrast, asdisclosed herein, separation of non-HDLs from HDLs can be achieved in asingle substantially uniform layer 94.

Further, it has been found that precipitation and separation take placein a direction that is substantially normal to the plane established bylayer 94. That is, while fluid movement occurs in all directions withinlayer 94, there is no significant net tangential migration of fluid fromone side of layer 94 to the other. Indeed, this embodiment does notincorporate or rely on different migration rates of plasma andprecipitated non-HDLs across layer 94. This is because fluid transportis through layer 94, not across it.

Many suitable materials can be used for layer 94, such as filter paperor cellulose acetate in combination with glass fibers. One suitablemembrane for layer 94 is CytoSep® grade 1660 membrane, 12.9 mils thick,available from Pall Corporation, East Hills, N.Y. Another suitablemembrane for layer 94 is paper grade 595, 0.180 mm (7.1 mil) thick,available from Schleicher & Schuell Bioscience, Inc., Keene, N.H.Further, layer 94 is substantially uniform throughout or symmetric. Thatis, while the matrix of layer 94 includes pores of different sizes, thematrix is consistent throughout the entire layer. Layer 94 isimpregnated with the solution described hereinbelow in the examples.

Total Cholesterol Measurement Stack

With further reference to FIG. 5, end stack 86 is spaced from middlestack 92 and is adjacent to and in fluid communication with layer 40.Stack 86 takes fluid from layer 40 and produces a colored response inreaction layer 90 that is proportional to the concentration of totalcholesterol in sample 58. Stack 86 also includes a blank or spacer layer88 whose main purpose is to maintain the relative thickness of allstacks approximately the same and, in so doing, improves overallcompression exerted upon matrix 36 by top portion 26 and bottom portion30 of strip holder 22. Blank layer 88 also retains residual blood cellspassed to it from layer 40. For purposes of this specification, the term“blank layer” refers to a layer such as layer 88 whose main purpose isto maintain all stacks at substantially the same thickness. Blank layer88 is not loaded with any reagents, but may be impregnated with awetting agent to improve fluid flow or may be impregnated with achromogen in applications wherein two test membranes are employed. Aspecific functioning example of a total cholesterol measuring stack 86is set forth in the Examples hereinbelow.

Triglycerides Stack

With further reference to FIG. 5, stack 98 is spaced from stack 92 andis adjacent to and in fluid communication with layer 40. Stack 98 takesplasma from layer 40 and produces a colored response in reaction layer102 that is proportional to the concentration of triglycerides in sample58. Stack 98 also includes a blank or spacer layer 100, that in thisembodiment is the same as blank layer 88. An example of a triglyceridesmeasuring stack 98 is set forth in the Examples hereinbelow.

It should be understood that once HDL concentration, total cholesterol,and triglycerides concentrations are determined from stacks 86, 92, and98, respectively, the concentration of LDL cholesterol can be calculatedby the well-known relationship:

LDL cholesterol=total cholesterol−triglycerides/5−HDL cholesterol.

A simple linear equation like that above can easily be programmed intothe instrument that optoelectronically reads the hybrid test strips,thus providing concentration of an additional analyte that was notmeasured directly.

Ampherometric Glucose

The ampherometric glucose test is performed by the electrochemicaltesting member 100. A mediator enables the testing of the glucose level.An enzyme such as glucoseoxidase (or Hexokinase or other enzyme specificfor glucose) may be used to create cuprous oxide. A voltage is appliedto the sample and the current is detected. The current detected isproportional to the concentration of glucose.

The use of ampherometric glucose detection systems may allow for theinterference of ascorbic acid, acetaminophen, and uric acid to beminimized. One approach for limiting these interferents is to utilize aselectively permeable membrane (permeable only to hydrogen peroxide, aresult of the enzymatic reaction). Another approach includes selectiveelectrocatalysis. Platinum and horserasdish peroxidase may be used forelectoreduction. These interferent limiting mechanisms are applicable totesting for other analytes and should not be construed as limited toglucose.

Examples of the layers that may form the test matrix are as follows:

EXAMPLE 1 Solution for Impregnation of Blood Separation Layer 40

The following impregnation solution was used:

Deionized water 800.00 mL D-Sorbitol 75.00 gm Sodium Chloride 10.00 gmAdjust the volume to 1 liter with deionized water.

EXAMPLE 2

Impregnation of Blood Separation Layer 40 with Solution of Example 1:

A fiberglass membrane (Ahlstrom Tuffglass™) 6.0″ (inches) wide wassubmersed in a re-circulating bath of the impregnation solution ofExample 1 at a rate of 0.5 ft/min. It then entered a tunnel of blowingwarm air (98° to 106° Fahrenheit) and low humidity (<5% relativehumidity (RH)) to completely dry. It was then slit into 0.80″ (inch)strips in preparation for assembly.

EXAMPLE 3

Impregnation of Blood Separation Layer 40 with Solution of Example 1:

A fiberglass membrane (Schleicher and Schuell 33) 6.0″ (inches) wide wassubmersed in a re-circulating bath of the impregnation solution ofExample 1 at a rate of 0.5 ft/min. It then entered a tunnel of blowingwarm air (98° to 106° Fahrenheit) and low humidity (<5% RH) tocompletely dry. It was then slit into 0.80″ (inch) strips in preparationfor assembly.

EXAMPLE 4 Solution for Impregnation of HDL Fractionation Membrane (Layer94):

The following impregnation solution was used:

Deionized water 800.00 mL Magnesium Sulfate 5.00 gm Phosphotungstic Acid45.00 gm Sorbitol 10.00 gm Adjust pH with NaOH or HCl pH 6.40-6.60Adjust the volume to 1 liter with deionized water.

EXAMPLE 5

Impregnation of Layer 94 with Solution of Example 4:

A synthetic fiber composite media (Pall CytoSep™ grade 1660) 12.9 (mils)thick, 5.90″ (inches) wide was submersed in a re-circulating bath ofimpregnation solution at a rate of 0.5 ft/min. It then entered a tunnelof blowing warm air (98° to 106° Fahrenheit) and a low humidity (<5% RH)to completely dry. It was then slit to 0.20″ (inch) strips inpreparation for assembly.

EXAMPLE 6

Impregnation of Layer 94 with Solution of Example 4:

A synthetic fiber composite media (Pall CytoSep™ grade 1661) 7.1 (mils)thick, 6.0″ (inches) wide was submersed in a re-circulating bath ofimpregnation solution at a rate of 0.5 ft/min. It then entered a tunnelof blowing warm air (98° to 106° Fahrenheit) and a low humidity (<5% RH)to completely dry. It was then slit to 0.20″ (inch) in preparation forassembly.

EXAMPLE 7

Impregnation of Layer 94 with Solution of Example 4:

A general purpose paper (Schleicher and Schuell 595) 6.0″ (inches) widewas submersed in a re-circulating bath of impregnation solution at arate of 0.5 ft/min. It then entered a tunnel of blowing warm air (98° to106° Fahrenheit) and a low humidity (<5% RH) to completely dry. It wasthen slit to 0.20″ (inch) strips in preparation for assembly.

EXAMPLE 8 Solution for Impregnation of Triglycerides Reaction Layer(Layer 102)

The following impregnation solution was used:

Deionized water 800.00 mL Triton X-100 1.00 gm CHAPS 0.70 gm KlucelCitrate Foundation 575.20 gm *see below 10% Gantrez AN139 20.80 gmCalcium Chloride, Anhydrous 0.20 gm Sucrose 25.20 gm Na2ATP 32.00 gmAdjust pH with NaOH or HCl pH 5.70 +/−0.10 MAOS 6.25 gm G3P Oxidase250.00 kU Peroxidase 750.00 kU Lipoprotein Lipase 625.00 kU GlycerolKinase 358.40 kU 4-amino antipyrine 5.55 g Deionized water 800.00 mLSodium Citrate 20.60 gm Citric Acid Monohydrate 6.30 gm MagnesiumChloride 1.43 gm BSA Std. Powder 20.00 gm Sodium Benzoate 2.0 gm KlucelEXF 10.00 gm Adjust pH to 5.5-5.7 Adjust the volume to 1 liter withdeionized water. *Klucel Citrate Foundation

EXAMPLE 9

Impregnation of Triglyceride Reaction Layer 102 with Solution of Example8:

A nylon membrane (Pall Biodyne A™) 0.4511 m pore size, 6.0″ (inches)wide was submersed in a re-circulating bath of impregnation solution ata rate of 1.0 ft/min. It then entered a tunnel of blowing warm air (98°to 106° Fahrenheit) and a low humidity (<5% RH) to completely dry. Itwas then slit to 0.20″ (inch) strips in preparation for assembly.

EXAMPLE 10 Solution for Impregnation of Cholesterol Reaction Layers(Layers 90 and 96)

The following solution was used to impregnate layers 90 and 96:

Deionized water 200.00 mL Triton X-100 0.77 gm Cholesterol Foundation532.00 gm *see below BSA Std. Powder 13.88 gm 10% Gantrez (w/v) 95.61 gmCHAPS 19.82 gm Sucrose 37.01 gm Adjust pH with NaOH or HCl pH 5.00+1-0.10 Potassium Ferocyanide 0.11 gm TOOS 0.37 gm MAOS 4.63 gmCholesterol Oxidase 74.00 kU Peroxidase 231.30 kU Cholesterol Esterase240.60 kU 4-Amino Anti-Pyrine 4.16 gm Adjust the pH if necessary to5.3-5.5 Adjust the volume to 1 liter with deionized water. Deionizedwater 800.00 mL Sodium Citrate Dihydrate 30.00 gm PVP K-30 60.00 gmBenzoic Acid 2.00 gm BSA Std. Powder 4.00 gm EDTA, disodium dehydrate1.47 gm Adjust pH with NaOH or HCL pH 5.40-5.60 Adjust the volume to 1liter with deionized water. Catalase 0.50 kU *Cholesterol FoundationNote: Even though the same impregnation solution is used for layers 90and 96, the result obtained in layer 90 is proportional to theconcentration of HDL cholesterol (since nonHDLs have been removed),whereas the result obtained in layer 96 is proportional to theconcentration of total cholesterol.

EXAMPLE 11 Impregnation of Cholesterol Reaction Layers (Layers 90 and96):

A nylon membrane (Pall Biodyne A™) 0.45 μm pore size, 6.0″ (inches) widewas submersed in a re-circulating bath of impregnation solution at arate of 1.0 ft/min. It then entered a tunnel of blowing warm air (98° to106° Fahrenheit) and a low humidity (<5% RH) to completely dry. It wasthen slit to 0.20″ (inch) in preparation for assembly.

EXAMPLE 12 Disbursement Layer 38:

A polyester membrane (Accuwik™, Pall Corporation) 13.0-14.0 mils thick,6.0″ (inches) wide is slit to 0.8″ wide and put on reels with a 3.0″core in preparation for assembly.

EXAMPLE 13 Disbursement Layer 38:

A polyester membrane (Accuflow Plus-P™, Schleicher & Schuell) 13.0-14.0mils thick, 6.0″ (inches) wide is slit to 0.8″ wide and put on reelswith a 3.0″ core in preparation for assembly.

EXAMPLE 14 Adhesive Layer 44:

A support material with adhesive (G&L 187) is slit to 0.8″ (inch) wide,then placed in a hole punching die to punch 3 (three) 0.140″ diameterholes, 0.215″ apart vertically and 0.378″ horizontally and put on reelswith a 3.0″ core in preparation for assembly.

EXAMPLE 15 Assembly of Test Matrix 36 and Holder 22:

All materials listed in examples 1-14 are placed upon a layering machinewhich consolidates the pre-slit membranes in a stacked format consistingof:

-   -   Disbursement layer 38    -   Blood Separation Layer 40    -   HDL fractionation layer 88    -   Untreated layers 94/100    -   Cholesterol Reaction Layers 90/96    -   Triglycerides Reaction Layer 102    -   Adhesive Layer 44

The hybrid test strip assembled as just described measuresconcentrations of HDL, total cholesterol, and triglycerides. Asdiscussed above, layers 90, 96, and 102 are aligned over holes 46 insupport layer 44, which in turn are aligned over openings 34 in thebottom portion 30 of hybrid test strip holder 22. A blue colorproportional to the concentration of the respective analyte can be seenin each of the respective openings 34.

It is envisioned that support layer 44 could be removed in commercialembodiments, as the support layer's function is to hold the other layersin place until the strips are assembled.

EXAMPLE 16 Calibration Curves

Several whole blood samples of known concentrations of HDL, totalcholesterol, and triglycerides were tested by:

-   -   1. Applying a 35-40 microliter sample to opening 32 of hybrid        test strips 20; and    -   2. Reading reflectance from the blue color on reaction layers        (as seen through 35 openings 34) on a portable whole blood        analyzer (BioScanner PIus™ instrument, Polymer Technology        Systems, Indianapolis, Ind.).

FIGS. 11-13 show calibration curves generated by plotting concentrationsof blood samples against percent reflectance (% R) values read on aBioScanner Plus™ instrument. FIGS. 14-16 show plots of measured HDL,total cholesterol, and triglycerides, respectively, versus knownconcentration. As shown, the coefficient of correlation R2 in each caseis very good.

The examples below illustrate construction of stacks for glucose andketones, which could be substituted for or added to the matrix 36described above.

EXAMPLE 17

The following structure was constructed as per Example 15 for a multipleanalytes hybrid test strip 20 that provides concentration of totalcholesterol, HDL cholesterol, and glucose:

-   -   Disbursement layer 38 (Accuflow Plus F™)    -   Blood Separation Layer 40 (Ahlstrom Tuffglass™)    -   HDL fractionation layer 88    -   Untreated layers 94/100 (layers 88, 94, and 100 all made of        CytoSep™ 1660)    -   Cholesterol Reaction Layers 90/96    -   Glucose Reaction Layer 102

Adhesive Layer 44 Solution for Impregnation of a Glucose Reaction Layer:

The following solution was used:

Deionized water 397.20 g Glucose foundation 537.30 g *See below Gantrez(10%) 19.40 g Potassium ferricynide 23.40 g Adjust the pH to 4.7 withcitric acid. MAOS 4.67 g Peroxidase 700.90 ku Glucose oxidase 467.20 ku4-amino antipyrine 4.21 g Adjust the pH to 4.7-4.9. Adjust the volume to1 liter with deionized water. Deionized water 800.00 g Triton x-100 1.86g Citric acid, monohydrate 4.00 g Sodium citrate, dehydrate 54.00 gPotassium EDTA 1.30 g PVP (40,000 daltons) 60.00 g Bovine serum albumin20.00 g Adjust the pH to 4.7-4.9 Catalase 50 U Adjust the volume to 1liter with deionized water. *Glucose foundation

EXAMPLE 18 Impregnation of Glucose Reaction Layer:

The process is the same as that used for the cholesterol reaction layers90 and 96 and triglycerides reaction layer 102. One suitable membranefor glucose reaction layer 102 is Thermopore™ from Pall Corporation.

EXAMPLE 19 Calibration Curves

Calibration curves for the three chemistries (Total cholesterol, HDLcholesterol, and glucose) were generated as in Example 16. Several wholeblood samples of known concentrations of HDL, total cholesterol, andglucose were tested by:

-   -   1. Applying a 35-40 microliter sample to opening 32 of hybrid        test strip 20; and    -   2. Reading reflectance from the blue color on reaction layers        (as seen through openings 34) on a portable whole blood analyzer        (CardioChek PA™ instrument, Polymer Technology Systems).

FIGS. 17-19 show calibration curves generated by plotting concentrationsof blood samples against percent reflectance (% R) values read on aCardioChek PA™ instrument.

EXAMPLE 20 Solution for Impregnation of a Ketone Reaction Layer:

The following solution was used:

Deionized water 493.78 g Igepal 660 0.99 g Ketone foundation 493.78 g*See below Sodium chloride 5.77 g Oxamic acid, sodium salt 0.55 gSucrose 24.69 g Adjust the pH to 7.9-8.1 NBT 5.46 g Diaphorase 264.67 kUHydroxybutyrate dehydrogenase 88.22 kU Deionized water 800.00 g Sodiumcitrate, dehydrate 24.00 g Bovine serum albumin 13.50 g PVP (30,000daltons) 50.00 g Adjust the pH to 7.9-8.1 Adjust the volume to 1 literwith deionized water. *Ketone foundation

EXAMPLE 21 Impregnation of Ketone Membrane:

The process and membrane are the same as those used for glucose membrane102 as described with reference to Example 18.

Test Method

A blood sample of approximately 30 to 50 microliters is contacted withthe center of the top surface of elongated disbursement layer 38 ofhybrid test strip 20. This is preferably performed by dispensing thesample from the tip of a micro pipette into application window 32. Theblood sample then spreads substantially throughout the entire length ofdisbursement layer 32. As a second step, although not necessarilysequential from the spreading step, the blood sample is delivereduniformly from substantially the entire length of the bottom surface ofdisbursement layer 38 to blood separation layer 40, which is believed toretain about 80% to 90% of the red blood cells. The fluid having about20% red blood cells remaining then is delivered to stacks 86, 92, and 98(FIG. 5). As the sample moves vertically through these stacks, blanklayers 88 and 100 retain any red blood cells that escape from layer 40,whereas layer 94 additionally precipitates and retains non-HDLcholesterol. Again, fluid moves through the stacks in a direction thatis substantially normal to the plane defined by the stacks. While fluidmovement is believed to be substantially completed within 10 to 20seconds, it takes longer for color to develop in layers 90, 96, and 100.In about two (2) minutes, color development at the bottom of each stackhas substantially reached an endpoint; and reflectance of each layer 90,96, and 100 may be measured and correlated with cholesterolconcentration as described above. Reflectance may be read andautomatically converted to concentration by available optoelectronicinstruments.

Since certain changes may be made in the above systems and methodswithout departing from the scope of the invention, it is intended thatall subject matter contained in the above description or shown in theaccompanying drawings may be interpreted as illustrative and not in alimiting sense.

1. A method of determining concentrations of a plurality of analytesfrom a single blood sample, placed in a single opening, the methodcomprising: (a) placing said single blood sample in an opening; (b)absorbing said blood sample with a test matrix that includes a pluralityof layers and a chromogenic agent; (c) generating a colored responsewith said test matrix wherein said colored response is proportional to aconcentration of a first analyte; (d) drawing a portion of said singleblood sample into a capillary tube; (e) contacting said portion of saidsingle blood sample with an electrode and a counter-electrode; and (f)measuring an electrical property of said single blood sample though saidelectrode and counter-electrode wherein said electrical property isproportional to a concentration of a second analyte in said single bloodsample.
 2. A method as in claim 1 wherein the drawing of (d) isaccomplished by a capillary effect of said capillary tube.
 3. A methodof determining concentrations of a plurality of analytes from a singleblood sample, placed in a single opening, the method comprising: (a)contacting said single blood sample with a top surface of an elongateddisbursement layer and spreading said single blood sample substantiallythroughout an entire length of said disbursement layer; (b) contactingsaid single blood sample with an end of a capillary tube such that aportion of said single blood sample is drawn into said capillary tube;(c) delivering said single blood sample from said disbursement layer toa first stack, a second stack, and a third stack, each of said first,second, and third stacks positioned adjacent to and in constant contactwith said disbursement layer, (d) moving the sample downward through thestacks in a direction substantially normal to the plane defined by thestacks; (e) producing a colored response at the bottom of each of thethree stacks, the colored response at the bottom of the first stackbeing proportional to the concentration of a first analyte in the bloodsample, the colored response at the bottom of the second stack beingproportional to the concentration of a second analyte in the bloodsample, and the colored response at the bottom of the third stack beingproportional to the concentration of a third analyte in the bloodsample; (f) delivering the portion of the single blood sample drawn intothe capillary tube to an electrode and a counter-electrode; and (g)measuring an electrical property of the single blood sample using theelectrode and counter-electrode wherein the electrical property isproportional to the concentration of a fourth analyte in the bloodsample.
 4. A method as in claim 3 wherein at least one of said stacksincludes a blank layer, said blank layer being a different layer thansaid disbursement layer, wherein said blank layer primarily functions tomaintain all stacks at substantially the same thickness.
 5. A method asin claim 3 wherein said first, second, and third analytes are TotalCholesterol, HDL Cholesterol, and Triglycerides, respectively.
 6. Amethod as in claim 3 wherein said fourth analyte is glucose.
 7. A methodas in claim 6 wherein a selectively permeable membrane is introducedbetween said electrode and counter-electrode in order to lesseninterferents.
 8. A method as in claim 6 wherein a selectiveelectrocatalyst is introduced in order to lessen interferents.
 9. Anapparatus for measuring concentration of multiple analytes in a wholeblood sample, comprising: (a) a test matrix comprising: (i) an elongatedporous disbursement layer; (ii) a blood separation layer adjacent to thebottom surface of said disbursement layer; and (iii) at least twovertically aligned stacks spaced apart and adjacent to the bottomsurface of said blood separation layer wherein a first one of saidvertically aligned stacks includes multiple layers, said multiple layersincluding a reagent and a chromagen; (b) an electrochemical testingmember comprising: (i) a capillary tube having a first and second end;(ii) an electrode; and (iii) a counter-electrode wherein said electrodeand counter-electrode are oriented in electrical communication with eachother when a blood sample is present; and (c) a hybrid test strip holderhaving a top portion and a bottom portion sandwiching said test matrixtherebetween; said top and bottom portions holding said electrochemicaltesting member; said top portion of said hybrid test strip holder havinga sample application window exposing a top surface of said disbursementlayer and said first end of said capillary tube; and said bottom portionof said hybrid test strip holder having at least one test reading windowthrough which bottom surfaces of said first and second stacks can beread.
 10. An apparatus as in claim 9 wherein said sample applicationwindow is positioned within a periphery defined by said stacks.
 11. Anapparatus as in claim 9 wherein said bottom surfaces of said stacks aresubstantially coplanar.
 12. An apparatus as in claim 9 wherein saidblood separation layer comprises a glass fiber matrix.
 13. An apparatusas in claim 9 wherein said electrochemical test panel further comprises:(iv) enzymatic reactants overlaying said electrode andcounter-electrode.
 14. An apparatus as in claim 9 wherein a selectivelypermeable membrane is introduced between said electrode andcounter-electrode in order to lessen interferents.
 15. An apparatus asin claim 9 wherein a selective electrocatalyst is introduced in order tolessen interferents.
 16. An apparatus as in claim 9 wherein saidelectrochemical testing member is interchangeable.
 17. A method ofcustomizing a hybrid test strip for determining concentrations of aplurality of analytes from a single blood sample, placed in a singleopening, the method comprising: (a) separating a top portion of saidhybrid test strip holder from a bottom portion of said hybrid test stripholder, said bottom portion of said hybrid test strip holder having arod; (b) placing an electrode chemical testing member on said bottomportion of said hybrid test strip holder such that a hole in saidelectrode chemical testing member is on said rod of said bottom portion;and (c) connecting said top portion of said hybrid test strip holder andsaid bottom portion of said hybrid test strip holder such that saidelectrochemical testing member is sandwiched therebetween; wherein saidhybrid test strip holder houses a test matrix comprising: (i) anelongated porous disbursement layer; (ii) a blood separation layeradjacent to said bottom surface of said disbursement layer; and (iii) atleast two vertically aligned stacks spaced apart and adjacent to saidbottom surface of said blood separation layer wherein a first one ofsaid vertically aligned stacks includes multiple layers, said multiplelayers including a reagent and a chromagen.